562
a potential surface with a shallow minimum. The repulsive part of this surface intersects an attractive potential surface and the crossing between the two surfaces is the rate-determining step of the reaction. A positive activation energy clearly corresponds to the crossing point lying at higher energies than the separated reactants. However, if the intersection lies below the separated reactants, an increase in temperature decreases the probability of crossing between surfaces due to the higher average velocity of the collision complex at the crossing point.l6 This is responsible for the negative temperature coefficient found in the reactions with tetramet hylet hylene. Acknowledgment. We thank the National Research Council of Canada for financial support. (16) V.N. Kondrat'ev, "Chemical Kinetics of Gas Reactions," Pergamon, London, 1964, p 217.
J. Connor, A. Van Roodselaar R. W. Fair, 0. P. Strausz* Department of Chemistry, University of Alberta Edmonton, Alberta, Canada Received September 24, 1970
Topography of Nucleic Acid Helices in Solutions. Steric Requirements for Intercalation' Sir: It is well known that planar molecules such as acridine orange, ethidium bromide, proflavine, and actinomycin may intercalate between base pairs in DNA.2 This
NO
I ?
A I
f
+
CH,CH,N(CH,),(CH,),I(CH,),
2Br-
h
a
1, R, = R, = H 2, R, = CHJ;Q2= H 3,R,=R2=CHd 4, R, = R2= CH,CH,
Figure 1, and the results of the low-shear viscometric studies are given in Table 11. A number of interesting observations may be made. (1) The reporter molecules 1 and 2 where Rl = R, = H and R1 = H, Rz = CH3, respectively, show a strong and a large induced hypochromic effect (47 and 41 circular dichroism ([e]' = -6590 and - 10,060) of the 4-nitroaniline transition on binding to salmon sperm DNA (Table I). ( 2 ) Considerable broadening of the pmr signal of the a- and b-methyl protons of 1 and 2 are observed in the DNA complex at 32°.4 In the case of DNA-2 complex the pmr signal of the c-methyl protons is completely broadened and indistinguishable from base-line noise at 32 and 5 8 " , indicating a restricted tumbling rate of the 4-nitroaniline ring of 2. At 90°, where melting of the DNA helix has occurred,j sharp resonance lines characteristic of the free reporter molecules are observed for the a-, b-, and c-methyl protons.
x),
Table I. Effect of Salmon Sperm DNA (ss DNA) on the Absorption and Induced Circular Dichroism Spectra of Reporter Molecules 1-4" ~
~
~~
~
~~~
~~~~
---.
Absorption spectra Reporter
1 2 3
4
ss DNA
-H,O-buffer--A""', nm 373 374 372 372
A""", 16,190 14,770 13,650 12,140
nm
389 393 375 374
emax
11,Ooo 10,450 13,540 11,500
--Circular
% Hb 47.0 41 .O 1 .o 5.6
dichroism-ss DNA----
7
At, nm 385 395 360-400 360400
(e)$ x
10-3
-6.59 -10.06 0.34 0.40
At 25.0 3= 0.2" in 0.01 M sodium phosphate buffer, pH 6.40-6.50 (0.01 M in Na+). Spectra were taken in 10" cells using Cary 14 and Cary 60 spectrometers. Values of Amax, P X , At, and (0)t in the presence of nucleic acid reported in this table are limiting values, Le., additional change in spectra is not observed at further excess of nucleic acid. Percentage hypochromicity H) = [eg;Jerx - 1.001100, where and c y are the extinction coefficients in the presence and absence of the polynucleotides.
(z
phenomenon leads to an increase in the length of the helix and is usually accompanied by an increase in the viscosity of the solution. In order to delineate the steric requirements for the intercalation process, reporter molecules, I, with increasing size of R substituents were synthesized. The results of the absorption and the induced circular dichroism studies of free and DNA-bound reporters are given in Table I. The temperature dependent partial proton magnetic resonance spectra are shown in (1) This is related (as XXII) to other articles on Topography of Nucleic Acids in Solutions. For part XXI, see E. J. Gabbay, B. Gaffney, and R. Glaser, Ann. N . Y . Acad. Sci., 171,810(1970). (2) (a) L. S. Lerman, J . Mol. Biol., 3, 18 (1961); (b) D. S. Drummond, N. J. Pritchard, V. F. W. Simpson-Gildemeister, and A. R. Peacocke, Biopolymers, 4,971 (1966); (c) G. Cohen and H. Eisenberg, ibid., 8, 45 (1969); (d) W.Muller and D. M. Crothers, J. Mol. Bid., 35, 251 (1968). (3) The reporter molecules 1-4 were prepared according to the procedure of E. J. Gabbay (J. Amer. Chem. Soc., 91, 5136 (1969)) and analyzed by ir, pmr, uv, and elemental analysis.
More rapid tumbling and/or efficient averaging of the chemical-shift environment is indicated. (3) The lowshear viscometric studies show that the intrinsic viscosity of the DNA-bound reporters 1 and 2 relative to free DNA are increased by 0.26 and 0.30, respectively (Table 11). The above results, 1, 2, and 3, are strongly suggestive that the 4-nitroaniline ring of reporters 1 and 2 is intercalated between base pairs in DNA. This is (4) If the rate of molecular tumbling of molecules in solutions is lower than the typical Larmor frequencies Wo (of the order of 108-109 the radians sec-1 for protons in the conventional magnetic field), then Tz, transverse relaxation time, is considerably diminished, leading to substantial line broadening of the proton signal (see 0. Jardetsky and C. D. Jardetsky in "Methods of Biochemical Analysis," D. Glick, Ed., Interscience, New York, N. Y.,1962). This situation is obtained if the proton is contaified in a rigid macromolecule, e.g., DNA, or if the proton is contained in a slowly tumbling small molecule bound to a macromolecule. (5) Evidence for the melting of DNA is seen in Figure 1 by the appearance of the thymine methyl peaks at 1.5-2.0 ppm [C.C. McDonald, W. D. Phillips, and J. Lazar, J . Amer. Chem. Soc., 89, 4166 (1967)l.
Journal of the American Chemical Society J 93:2 J January 27, 1971
563
a
I
NO2
I I1
NO2
I at 90'
I MJn.! at 90"
DNA.2 at 58'
n
n I I I
4.0
I I I I I l l l I I I I I I I I I I I I / l l l / l l l l l l l l l l l l l
3.0
2.0
I .o
0.0
d at 90"
j DNA 4 at go"
DNA.3 at 32'
Figure 1, The temperature-dependent partial pmr spectra of free and DNA-bound reporter molecules: (a) reporter 1; (b) reporter 2; (c) reporter 3; (d) reporter 4. Spectra were taken on a Varian A-60A. Sonicated low molecular weight ss D N A was used at 0.16 mol of PA. in DzO in lo-' sodium phosphate buffer (pD 7.0 f 0.2). The concentration of the reporter molecule was 0.02 M.
in contrast to the behavior of reporter molecules 3 and 4 with N,N-dimethyl- and N,N-diethyl-substituted 2-car-
boxyamido groups, respectively. For example, the latter reporters show little or no effect on the absorption Communications to the Editor
564 Table 11. Relative Intrinsic Viscosity of DNA-Reporter Complexes t o Free DNAa,* Reporter
(d/(tlO)
Health Service and GB-16044 from the National Science Foundation. (9) Recipient of a U. S. Public Health Service Career Development Award from the National Institutes of Health. (10) Predoctoral Fellow (1968-present), Rutgers, The State University, New Brunswick, N. J.
Edmond J. Gabbay,*9 Ameli DePaolislO
The intrinsic viscosities of free DNA, (qo), and the DNAreporter complex, ( q ) , were calculated using the equation qsp/c = (7) k'(+c and assuming that the value of the Huggins constant, k', is the same for D N A and the DNA-reporter complex. This assumption is justifiable and has been recently shown by P. D. Ross and R. L. Scruggs [Biopolymers, 2,79 (1964)l to apply for D N A and DNA-dye complexes under similar ionic strength conditions. *Viscosity measurements were carried out using 1.13 X 10-3 M in poise/liter of D N A and 1.0 X M in reporter molecule in 0.025 M sodium phosphate buffer (pH 6.50) at 25" using a Zimm viscometer. Under these conditions, the reporter molecules are fully bound to D N A as evidenced by spectrophotometric titration studies.
Department of Chemistry, University of Florida Gainesuille, Florida 32601 Received October 19, 1970
+
and induced circular dichroism of the 4-nitroaniline transition on binding to DNA (Table I). In addition, the pmr signals of the a-, b-, and c-alkyl protons of 3 and 4 are clearly discernible in the presence of DNA at 32" with only a slight broadening and an upfield chemical shift.6 In line with these results, the intrinsic viscosity of the DNA-bound reporters 3 and 4 is slightly decreased (Table 11). A similar decrease of the intrinsic viscosity of DNA is observed with the diammonium The mode of salt 5 , (CH3)3N+(CH2)3N+(CH3)3.2Br-. binding of 3 and 4 to DNA seems to be external electrostatic binding to the phosphodiester chain with the 4-nitroaniline ring freely tumbling in solution. Thus, intercalation between base pairs of DNA by 3 and 4 is inhibited presumably due to either (a) the absence of an H-bond donor or (b) steric hindrance by the nonplanar N,N-dialkylcarboxamido substituent of 3 and 4. We favor the latter alternative since the requirement for a hydrogen bond donor at the 2 position of the 4-nitroaniline ring is not essential for the intercalation process. For example, it has been shown that the unsubstituted, the 2-cyano-, and the 2-nitro-substituted 4-nitroaniline labeled diamines intercalate between base pairs in DNA as evidenced by the absorption, induced circular dichroism, proton magnetic resonance, and viscosity studies.' It should be noted that the results of the preliminary binding studies of these reporters to DNA show that the reporter molecules 1 and 2 are bound approximately 10-15-fold more strongly than 3 and 4.* This is in line with the proposed binding modes of these molecules described above. Further work along this area is in progress. Acknowledgment. This work was supported by Grants GM17503 and GR118653 from the U. S . Public (6) It should be noted that the alkyl protons of the N,N-dialkylcarboxamido group of 3 and 4 are not equivalent at 32" due to the restricted rotation about the amide group. At go", however, coalescence of the pmr signals is obtained (Figure IC and d). (7) (a) F. Passero, E. Gabbay, B. L. Gaffney, and T. Kurucsev, Macromolecules, 3, 158 (1970); (b) E. Gabbay, J . Amer. Chem. Soc., 91, 5136 (1969); (c) €3. L Gaffney, Ph.D. Thesis, Rutgers, 1969. (8) Binding data were obtained by competitive binding studies using a cation exchange resin technique: A. DePaolis, Ph.D. Thesis, Rutgers, 1971. Journal of the American Chemical Society
Some Novel Reactions of Vinyl Isocyanide with Organophosphorus and Organoarsenic Compounds
Sir: Recently the base-catalyzed addition of phosphorushydrogen bonds across the vinyl double bonds of vinylphosphines to give polytertiary phosphines with PCH2CHzP units was reported.' This communication reports base-catalyzed additions of phosphorus-hydrogen bonds to vinyl isocyanide2 which provide routes not only to a phosphine isocyanide with a PCH,CH,NC unit but also to the first derivative of the 3-azaphosphole heterocyclic system. Reaction of diphenylphosphine with excess vinyl isocyanide in boiling benzene for -20 hr in the presence potassium tert-butoxide catalyst resulted in of -10 addition of the phosphorus-hydrogen bond across the vinyl double bond to give a 53 % yield of slightly yellow viscous liquid, (C6H&PCHzCHzNC: bp 139-142 O (0.05 mm) (Anal. Calcd for C15H14NP: C, 75.4; H, 5.9; N, 5.9; P, 12.9; mol wt, 239. Found: C, 75.1, 75.7; H, 6.2, 5.7; N, 5.6, 5.3; P, 13.1; mol wt, 238 (osmometer in benzene solution)). The infrared spectrum of neat (C6H&PCHZCH2NC exhibited a strong vCN frequency at 2154 cm-' confirming the presence of an isocyanide group. The proton nmr spectrum of (C6H&PCHzCH2NC in CDC& exhibited the usual aromatic proton resonance at 7 2.74 as well as two resonances at 6.72 (quartet, 8-Hz separation) and 7.66 (triplet, 8-Hz separation) arising from the nonequivalent methylene groups. The mass spectrum of (C6HJzPCHzCH2NCdid not exhibit a molecular ion. Instead the highest m/e ion was (C6H&PCzH3+ formed by elimination of HCN from the molecular ion. Other ions frequently found in the mass spectra of (C6H&PR derivatives314 were also observed (e.g., (CsHb)zPH+, ClzHsPf, C1zHlo+, C12H8+, C8H?pf, c8H6P+, C6H8Pf, C&P+, and C6H4P+). The arsenic analog (C6H6)2AsCH2CHzNC, a liquid, bp 153O (2 mm), was similarly prepared in 3 1% yield from diphenylarsine and excess vinyl isocyanide and characterized by elemental analyses, molecular weight determination, and infrared and proton nmr spectra. The compounds (C6H&ECHzCH2NC (E = P or As) are the first known compounds containing both an isocyanide group and a tertiary phosphorus or arsenic atom. All of these functionalities coordinate with transition metals to form extensive series of com(1) R. B. King and P. N. Kapoor, J . Amer. Chem. Soc., 91, 5191 (1969). (2) D. S. Matteson and R. A. Bailey, ibid., 90,3761 (1968). (3) D. H. Williams, R. S. Ward, and R. G. Cooks, ibid., 90, 966 (1968). (4) R. Colton and Q.H. Porter, A u s f . J . Chem., 21, 2215 (1968).
93:2 / January 27, 1971
